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Abstract

Background

Circadian clocks are internal daily time keeping mechanisms that allow organisms to anticipate daily changes in their environment and to organize their behavior and physiology in a coherent schedule. Although circadian clocks use temperature compensation mechanisms to maintain the same pace over a range of temperatures, they are also capable of synchronizing to daily temperature cycles. This study identifies key properties of this process.

Results

Gradually ramping daily temperature cycles are shown here to synchronize behavioral and molecular daily rhythms in Drosophila with a remarkable efficiency. Entrainment to daily temperature gradients of amplitudes as low as 4°C persisted even in the context of environmental profiles that also included continuous gradual increases or decreases in absolute temperature. To determine which elements of daily temperature gradients acted as the key determinants of circadian activity phase, comparative analyses of daily temperature gradients with different wave forms were performed. The phases of ascending and descending temperature acted together as key determinants of entrained circadian phase. In addition, circadian phase was found to be modulated by the relative temperature of release into free running conditions. Release at or close to the trough temperature of entrainment consistently resulted in phase advances. Re-entrainment to daily temperature gradients after large phase shifts occurred relatively slowly and required several cycles, allowing flies to selectively respond to periodic rather than anecdotal signals. The temperature-entrained phase relationship between clock gene expression rhythms and locomotor activity rhythms strongly resembled that previously observed for light entrainment. Moreover, daily temperature gradient and light/dark entrainment reinforced each other if the phases of ascending and descending temperature were in their natural alignment with the light and dark phases, respectively.

Conclusion

The present study systematically examined the entrainment of clock-controlled behavior to daily environmental temperature gradients. As a result, a number of key properties of circadian temperature entrainment were identified. Collectively, these properties represent a circadian temperature entrainment mechanism that is optimized in its ability to detect the time-of-day information encoded in natural environmental temperature profiles. The molecular events synchronized to the daily phases of ascending and descending temperature are expected to play an important role in the mechanism of circadian entrainment to daily temperature cycles.

Background

Due to the rotation of our planet around its own axis most life forms on earth are exposed to daily rhythms in environmental light and temperature. In response, internal daily biological timekeepers, termed circadian clocks, have evolved. These circadian clocks provide organisms with the ability to reliably predict regular daily changes in their environment and to organize their bodily functions and behavior in a coherent daily schedule [1-6]. The selective advantage of possessing a circadian clock that is tuned to environmental rhythms has been directly demonstrated by experiments using cyanobacteria [7] and Arabidopsis [8,9]. The defining properties of circadian clocks include (1) ~24-h (circadian) periodicity, (2) autonomous time keeping under constant conditions, (3) entrainment to environmental time cues such as light or temperature, (4) control of overt biological rhythms, and (5) maintenance of a constant pace over a range of environmental temperatures [1-6]. The molecular circadian clocks that have been described in higher eukaryotes all have transcriptional feedback circuits that allow them to create self-sustaining molecular oscillations in gene expression. In the Drosophila clock the positive transcriptional regulator CLOCK/CYCLE (CLK/CYC) induces peak transcript levels of a number of clock-controlled genes, including period (per), timeless (tim), vrille (vri) and PAR-domain protein 1 (Pdp1). Subsequent accumulation and nuclear entry of PER and TIM proteins results in direct inhibition of CLK/CYC, whereas VRI acts as a negative transcriptional regulator for the Clk promoter and PDP1 functions as a transcriptional activator. Elaborate post-translational mechanisms are at work to ensure that gene expression oscillations are produced with a stable circadian period length.

While the molecular mechanisms underlying circadian time keeping and synchronization to environmental light/dark cycles are relatively well understood, this is not yet the case for temperature entrainment. Circadian clocks have a complex relationship with environmental temperature, showing the ability to synchronize to daily temperature cycles as well as adjust their daily phase to seasonal differences in average temperature while retaining the ability to run at the same speed over a wide range of average daily temperatures [1,4,10,11]. Repeated daily temperature cycles can entrain clocks, while single temperature cycles, pulses, or steps can reset circadian phase.

Prior to the present study, it was, however, not known what specific features of daily temperature profiles determined circadian phase and how those phase determinants were interpreted in the context of complex environmental profiles that better resemble natural conditions. The analyses outlined below methodically addressed these issues and indicated that the Drosophila circadian clock is remarkably sensitive to the environmental daily time cues represented in natural environmental temperature profiles.

Results and discussion

To determine whether the Drosophila clock could be synchronized to daily cycles of gradual temperature changes circadian entrainment of locomotor behavior was examined during and after exposure to daily temperature gradients in constant dark (DD) conditions (see Additional file 1). An example of the strong synchronizing effect of daily temperature gradients is represented in Figure ​Figure1.1. Flies were exposed to a gradual 18°C to 25°C temperature cycle and then released at constant 25°C. The maximal rate of temperature change during entrainment was only ~0.8°C/h, yet the phase of locomotor activity for both male and female flies was efficiently synchronized to coincide with the time of day corresponding to the temperature maximum. This phase relationship persisted during free running conditions indicating that the synchronized behavior represented a synchronized circadian clock rather than a purely temperature-driven direct effect on behavior.

Efficient entrainment of circadian behavior to daily temperature gradients. Twelve female and sixteen male Canton-S flies were analyzed individually for synchronization of locomotor activity during and after exposure to a daily temperature gradient in...

Analyses of temperature-entrained locomotor behavior such as those shown in Figure ​Figure11 suggested that the entrained activity peak phase was determined by the phase of the temperature maximum. This hypothesis was systematically tested by comparing the peak phase of synchronized locomotor activity with that of the entraining daily temperature gradient for eight different temperature protocols varying in amplitude (between 7°C and 12°C), peak phase, absolute temperature (from between 14°C and 22.5°C to between 22°C and 30°C), relative duration of thermophase, cryophase, temperature ascent or descent (see Z1-8 in Additional file 2 and Additional file 3). These protocols represent a diverse set of daily temperature profiles in which the duration of the temperature peak and trough vary from a discrete point in time to 15 h and the phases of ascending and descending temperature may be as short as 4 h or as long as 12 to 15 h. The results of this analysis are summarized in Figure ​Figure2A2A and ​and2B.2B. Most of the variance in peak activity phase is explained by a linear relationship with the time of peak temperature (see Figure ​Figure2A2A and ​and2B,2B, and Additional file 3). Moreover, this relationship is relatively insensitive to changes in the time of trough temperature or other features of the temperature profile (see Figure ​Figure2B2B and ​and2C).2C). Peak temperature also appears to be the predominant determinant of the phase of activity offset (see Figure ​Figure2C,2C, and Additional file 3) which shows tight linkage to the time of peak activity. When a broad temperature peak is used, peak and offset of locomotor activity appear to synchronize to its midpoint (see Figure ​Figure2C,2C, and Additional file 3).

Daily peak temperature is tracked by daily peak locomotor activity. Locomotor activity was recorded for Canton-S flies subjected to one of eight different daily temperature gradients in constant darkness (Z1-8 as indicated in Additional file 2 and Additional...

In addition, other features of the activity profile do change as a result of a prolonged thermophase. In particular, activity profiles tend to assume a bimodal character with distinct morning and evening peaks. In this context, the evening activity peak shows the same phase relationship to the temperature maximum as is observed for the peak of unimodal activity profiles, while the morning activity peak occurs shortly after the temperature trough, well in advance of peak temperature. One possible interpretation of these observations is that both the phase of temperature ascent and temperature descent regulate the timing of evening activity. To further explore this possibility a 48-h temperature cycle was used that results from modifying a daily temperature gradient by extending the duration of the temperature peak by 24 h (see Figure ​Figure2D).2D). As a result, the temperature protocol alternates entrainment days with defined phases for temperature trough, ascent and descent with days of free run at constant peak temperature. If, in fact, the mid-point of the temperature peak acted directly as the main phase determinant for entrainment, this 48-h protocol would contain conflicting signals because its mid-point of peak temperature coincides with the same time of day as its (subjective) temperature trough. However, flies showed efficient entrainment to this protocol with daily peak activity consistently occurring between the phases of (subjective) temperature ascent and descent, suggesting that the combination of these two signals acts as the phase determinant. Given the observation, noted above, that the phase of entrained locomotor activity is relatively insensitive to the shape of the daily temperature profile, the relative timing of temperature ascent and descent can apparently be detected efficiently across the different temperature protocols. Taken together, these results indicate that the phase of peak unimodal or evening activity is synchronized to coincide with the daily temperature peak by the combined action of temperature increases and decreases.

Although temperature affects daily behavior via synchronization of the circadian clock, as discussed above, it also modulates behavior via clock-independent mechanisms. The potential involvement of clock-independent temperature responses in shaping the daily locomotor behavior profiles observed in association with daily temperature gradients was studied in flies carrying arrhythmic clock gene alleles. The daily locomotor activity profiles of null mutants for period (per01) [12], timeless (tim01) [13], and cycle (cyc01) [14] were determined in the presence of a daily temperature gradient and compared with wild-type controls in a similar genetic background (y w). The behavioral profiles observed for arrhythmic mutant flies (see Figure ​Figure3)3) suggest that, in response to a daily temperature gradient, a functional circadian clock is required for anticipation of daily temperature changes, but not for the generation of diurnal activity rhythms per se. The temperature-driven behavior of per01 and tim01 mutants resembled the temperature-entrained behavior of wild-type controls in terms of the phase of peak and offset of activity, whereas the temperature-driven locomotor activity of cyc01 mutant females showed a similar peak, but somewhat delayed activity offset. The daily activity profile of cyc01 mutants appeared to track the phase of descending temperature more closely with approximately half of the total activity occurring during the phase of temperature descent in both males and females, suggesting that this aspect of temperature-dependent behavior may be modulated by CYC function in per01 and tim01 flies even in the absence of a self-sustaining oscillator. As expected, the absence of clock function in per01, tim01, and cyc01 mutants was evident in their lack of rhythmic behavior under constant conditions and their immediate resynchronization to phase-shifted temperature gradients (data not shown). In addition, the arrhythmic mutants showed a unimodal daily pattern of activity, whereas the wild-type control flies exhibited a bimodal profile that included a morning activity peak and a subsequent period of rest in advance of the evening activity peak associated with the temperature maximum. Thus, a functional circadian clock appears to be required to generate a distinctly bimodal daily activity profile in the context of a simple daily temperature gradient. This observation is in agreement with the previously documented clock-dependent modulation of midday rest or 'siesta', which is a feature of wild-type behavior that is influenced by gender, genetic background, and the environmental protocol of temperature and light [15-17]. In this context, it is worth noting that Canton-S flies in the presence of the same temperature gradient did not reproduce the bimodal activity profile of y w flies (see Figure ​Figure3;3; data not shown), while they did exhibit bimodal temperature-entrained activity in response to a different temperature gradient (cf. Figure ​Figure2C2C).

The phase of temperature-entrained circadian behavior is determined by the synchronizing daily temperature gradient as well as the relative temperature of release into free run

Given the strong clock-independent regulation of daily locomotor activity by temperature gradients it was important to determine whether the phase relationship between peak temperature and peak activity was maintained under constant conditions. Drosophila maintains molecular and behavioral circadian rhythmicity over a wide range of temperatures including those used in the entrainment protocols in this study. Circadian activity profiles were determined following entrainment to one of the eight daily temperature profiles described above (Z1-8 in Additional file 2 and Additional file 3). A total of fourteen different protocols resulted from combination of these eight temperature gradients with subsequent release at different times and temperatures (see Additional files 2 and 3). As predicted based on diurnal temperature-synchronized behavior the timing of peak temperature during entrainment was found to be a key determinant of the phase of peak and offset of circadian activity. However, about half of the temperature protocols showed additional modulation of the phase of circadian activity resulting in an advance relative to the time of the subjective temperature peak by 3 to 7 h. This effect was found to be largely determined by the relationship between the free running temperature and the temperature range of prior entrainment and is summarized in Figure ​Figure4A4A and illustrated by some examples in Figure ​Figure4B.4B. When flies are released from a daily temperature gradient into free run at peak temperature their activity peak and offset are maintained at essentially the same phase as during entrainment. This principle was not obviously affected by variations in amplitude, peak phase, absolute temperature, and relative duration of thermophase, cryophase, temperature ascent or descent among the different entraining temperature gradients. In contrast, release from entrainment at the temperature trough consistently resulted in a phase advance of the peak and offset of circadian activity. The behavioral phases of flies released at peak versus trough temperatures showed non-overlapping distributions with a highly significant difference (t-test P < 10-6). Additional data from flies released at intermediate temperatures is consistent with a roughly linear relationship between the relative release temperature and the resulting phase advance.

The phase of temperature-entrained circadian behavior is modulated by the relative temperature of release into free run. Locomotor activity was recorded for 28 groups of Canton-S flies (14 of each gender) subjected to 1 of 12 different combinations of...

In nature, flies encounter daily temperature gradients in the presence of a light/dark cycle. Based on the natural occurrence of rising temperatures during the day and falling temperatures during the night, light and temperature entrainment were predicted to be roughly in phase if light onset and offset occurred at the same time of day as trough and peak temperature, respectively. Indeed, the circadian phase resulting from light entrainment was essentially unaltered after subsequent exposure to an in-phase daily temperature gradient and vice versa (see Figure ​Figure5A,5A, left-hand diagrams). In contrast, when flies were sequentially exposed to oppositely aligned daily light/dark cycles and temperature gradients, circadian activity was shifted to the opposite phase during and following the second entrainment condition (see Figure ​Figure5A,5A, right-hand diagrams). Exposure to simultaneous in-phase daily light/dark and temperature entrainment resulted in a diurnal behavior profile identical to that observed for light/dark entrainment with respect to evening activity, whereas morning activity rose more gradually upon superposition of the temperature gradient (see Figure ​Figure5B).5B). The acute increase in morning activity observed in response to the sudden onset of bright light at constant temperature has been dubbed 'startle response' and does not depend on a functional clock [18,19]. Modulation of this aspect of morning activity by a daily temperature gradient is, therefore, not necessarily associated with a change in clock-dependent regulation. The circadian locomotor activity profile resulting after simultaneous in phase light and temperature entrainment was essentially the same as that separately observed after either type of entrainment (data not shown). Introduction of an oppositely aligned daily temperature gradient in the presence of a light/dark cycle resulted in a dramatic advancement (~5 to 6 h) of the onset of evening activity (see Figure ​Figure5C).5C). This effect apparently required interaction between the light- and temperature-dependent entrainment mechanisms, because it produced an increase in activity at a time of day when neither light nor temperature elicited this effect on their own (see also wild-type data in Figure ​Figure3;3; data not shown). Moreover, when arrhythmic mutant flies were exposed to the same combined light and temperature protocol they did not show a consistent increase in activity in anticipation of the lights-off transition (see Additional file 4). In summary, these results indicate that the natural alignment of light and temperature is expected to produce cooperative entrainment of daily locomotor activity.

Synchronization of circadian behavior to daily temperature gradients proceeds in a cumulative and relatively slow manner

In comparison with light/dark cycles daily temperature gradients provide weaker entrainment signals. While a single light/dark cycle is often sufficient to cause large phase shifts and near-complete entrainment [19,20] a single daily temperature gradient has more limited phase-resetting potential. This principle is illustrated by the experiments represented in Figure 6A, B and ​and6C.6C. Light-entrained flies were exposed to zero, one, three, or five anti-phase daily temperature gradients and then released into free run. Comparison of the resulting circadian activity profiles (Figure ​(Figure6B6B and ​and6C)6C) indicated that cumulative phase advances resulted from exposure to repeated daily temperature gradients (see Figure ​Figure6A).6A). Phase resetting was essentially complete after five days of entrainment as indicated by the circadian activity profiles resulting from prolonged exposure to the same temperature gradient (cf. Figure ​Figure5A5A and Figure ​Figure6B6B and ​and6C6C).

Phase resetting responses to daily temperature gradients are relatively slow and cumulative. After entrainment to 12-h dark/12-h light cycles (lights on from noon to midnight at constant 25°C for ≥4 days) groups of Canton-S male and female...

Flies selectively synchronize their circadian behavior to daily temperature gradients even in the presence of continuous temperature increases or decreases

The temperature profiles experienced by flies in nature vary not only in a daily fashion but also due to seasonal influences and to less predictable short-term factors such as the weather and the location of the animal. Daily temperature gradients will, therefore, represent reliable and consistent synchronizing signals for daily molecular and behavioral rhythms only if they can be selectively recognized in the presence of other temperature signals. A simple test of this principle was carried out by combining daily temperature gradients with a 0°C, 1.5°C, or 4°C daily range with continuous increases or decreases (3°C per day) in absolute temperature (see Additional file 5). At the start of the experiment daily locomotor activity was entrained to selected phases with the help of light/dark cycles. Next, flies were exposed for four days to one of the six temperature protocols described above before release at constant temperature (see Figure ​Figure7).7). The resulting peak and offset of subsequent circadian activity were determined and compared for different phase relationships with the initial light/dark cycle. In most cases phase shifts relative to the original light-entrained evening activity peak were observed, but for the 0°C and 1.5°C daily temperature gradients these shifts were largely independent from the phase relationship with the previous light/dark cycle. Such constitutive phase changes do not result from entrainment to a daily temperature component, but rather from the effects of continuous increases or decreases in absolute temperature on activity. In contrast, phase-dependent resetting was consistently observed for protocols involving the 4°C daily temperature gradient (cf. data for zigzag versus step and ramp protocols in Figure 8A, B, C and ​and8D).8D). This is nicely illustrated in a direct comparison of the circadian activity profiles resulting from exposure to simple continuous temperature increases or decreases with or without the 4°C daily temperature gradient (Figure ​(Figure8B8B and ​and8D8D).

Environmental protocols used to study temperature entrainment in the context of continuous increases or decreases in absolute temperature. Different environmental protocols were used as indicated. First, flies were entrained to three 12-h light/12-h dark...

Circadian behavior can selectively synchronize to daily temperature gradients that are combined with continuous increases or decreases in absolute temperature. Median activity records for each experimental condition described in Figure 7 were used to...

Entrainment of molecular rhythms to daily temperature gradients

The phases of circadian gene expression resulting from entrainment to a daily temperature gradient can be predicted based on the observed alignment of molecular and behavioral circadian rhythms during and after entrainment to light/dark cycles [21]. Thus, peak expression of CLK/CYC-regulated transcripts such as per, tim, and vri, which coincides with the light-entrained evening locomotor activity peak at dusk, is expected to occur during the temperature-entrained evening activity peak, which falls near the temperature maximum of a synchronizing daily temperature gradient. To validate these predictions per, tim, and vri transcript rhythms were determined in adult head extracts during and after entrainment to a daily temperature gradient. Indeed, CLK/CYC-regulated transcript profiles show the same phase relationship to the evening locomotor activity peak after entrainment to daily temperature gradients (see Figure ​Figure9A)9A) as previously demonstrated for light/dark cycles [20].

Clock gene expression rhythms during and after entrainment to daily temperature gradients. Canton-S flies were synchronized to a temperature gradient (16°C to 28°C) in constant darkness and then, during the experimental day, either kept...

As part of these experiments the effect of different relative release temperatures was examined as well. During the first full day of free running conditions no obvious phase changes were observed in per, tim, or vri transcript rhythms regardless of the relative release temperature (see Figure ​Figure9A).9A). However, the absolute levels of tim transcript were reduced upon release at relatively cold temperatures. Temperature-entrained PER and TIM protein phases maintained a normal delay (~6 h) relative to transcript levels both during exposure to the daily temperature gradient and during subsequent free run at constant 17°C (see Figure ​Figure9B9B).

Conclusion

The present study systematically examined the entrainment of clock-controlled behavior to daily environmental temperature gradients. As a result, a number of key properties of circadian temperature entrainment were identified. Collectively, these properties, as described below, represent a circadian temperature entrainment mechanism that is optimized in its ability to detect the time-of-day information encoded in natural environmental temperature profiles.

First, daily temperature gradients show a remarkable efficiency at entraining behavioral and molecular daily rhythms in Drosophila. The minimal range identified here for efficient entrainment by temperature gradients (1.5 to 4°C) is similar to that previously reported for square wave temperature cycles (2 to 3°C; [19,22]). Temperature input pathways, therefore, appear to be more than sensitive enough to respond to a wide variety of daily environmental temperature profiles.

Second, synchronization of circadian locomotor behavior is associated with detection of changes in relative temperature rather than absolute temperature. Circadian phase showed comparatively little dependence on absolute temperature, but was, instead, reliably predicted based on the phase of the relative temperature peak and the relative temperature of release. The use of a diverse panel of daily temperature wave forms and associated release temperatures in this study allowed this property of circadian temperature entrainment to be recognized. A crucial benefit of entrainment to relative temperature changes is that circadian clocks can effectively respond to daily temperature oscillations in the presence of seasonal (or other) changes in average temperature.

Third, the daily temperature peak as defined by the alignment of both ascending and descending daily temperature phases sets the circadian phase of entrainment. Peak circadian locomotor activity was strongly linked to the configuration of the ascending and descending temperature phases during entrainment. And this association was maintained irrespective of the phase of other features of the synchronizing daily temperature profile such as the trough. Again, analysis of a diverse panel of daily temperature wave forms in the present study was instrumental in allowing this property to be identified. A combined role of temperature increases and decreases in the circadian entrainment of eclosion in Drosophila pseudoobscura populations was proposed in an early study by Zimmerman et al. [23]. It is, therefore, very well possible that circadian temperature entrainment of locomotor activity and eclosion make use of the same mechanism.

Fourth, the relative temperature of release into free run modulates circadian phase. Whereas release into free running conditions at a temperature matching the entrainment peak has no apparent effect on circadian phase, progressive phase advances result from release at temperatures approaching the entrainment trough. Thus, changes in average daily temperature modulate rather than block entrainment to daily temperature gradients. This arrangement may allow for the simultaneous integration of both seasonal and daily information in generating rhythmic behavior. Seasonal modulation of circadian behavior occurs in part as a result of temperature sensitive splicing of the last intron of the clock gene period [16]. Indeed, lower temperatures directly induce more efficient splicing and, as a result, an advanced molecular and behavioral phase [17,24]. Although temperature-dependent splicing of per may have contributed to the phase advances observed upon release at lower absolute temperatures this mechanism cannot fully account for the observed sensitivity to relative temperature changes. Behavioral phase advances were observed upon release at constant trough temperature even for mutants that block per splicing (see Additional file 6). Moreover, no obvious molecular phase advance in per transcript or protein accumulation was detected for wild-type flies released at low relative temperature (see Figure ​Figure9A9A and ​and9B,9B, above).

Fifth, circadian phase responses to daily temperature profiles occur slowly and often require several days to be completed. Daily temperature gradients are weaker synchronizers of circadian behavior than light/dark cycles and have a more limited phase shifting potential per cycle. As a result, large temperature-dependent phase shifts are not triggered by single, anecdotal fluctuations in temperature, but rather by repeated exposure to convergent phase-shifting signals over the course of a number of days. The Drosophila clock is, therefore, tuned to repeated daily temperature components and able to resist dramatic short-term responses to irregular fluctuations in environmental temperature. Given the complexity and irregularity of natural environmental temperature profiles, this appears to be a very useful adaptation. The temperature-dependent phase responses described here are comparable to those reported earlier for square-wave temperature pulses and cycles [21,25,26]. For example, Boothroyd and colleagues found that it took flies 5 days to complete a 10-h phase advance in response to a square wave 18°C/25°C temperature cycle, whereas Busza et al. observed that 8-h phase advances and 8-h delays in response to a 20°C/29°C square wave were achieved over a time frame of more than 6 days or 5 days, respectively [21,25].

Sixth, daily cycles in relative temperature can synchronize circadian behavior even when they are presented as part of more complex temperature profiles. Entrainment to daily temperature gradients persisted even in the context of continuous gradual temperature increases or decreases. To effectively detect the time-of-day information encoded in environmental temperature profiles circadian clocks have to be able to sense daily changes in relative temperature in the context of patterns that contain unrelated thermal signals, for example, due to weather-related developments. The present study shows that this is, indeed, the case in examples where continuous changes in absolute temperature are used as a back-drop for entrainment.

Seventh, naturally aligned daily temperature and light cycles provide cooperative circadian entrainment. Daily temperature gradient and light/dark entrainment reinforced each other if the phases of ascending and descending temperature were in their natural alignment with the light and dark phases, respectively. In nature, entrainment of the Drosophila circadian clock to temperature profiles occurs in the context of a daily light/dark cycle. To achieve the highest sensitivity towards environmental time-of-day information the circadian clock is expected to show light/dark-entrained and temperature-entrained phases that approximately coincide when these zeitgebers are in their natural alignment. The phase determinants of temperature and light entrainment both result from changes in solar irradiance and normally occur in succession during the second half of the day. Peak temperature, the main synchronizing signal for thermal entrainment (see above), generally falls in the afternoon several hours in advance of dusk [27], which is the major phase determinant of light entrainment (for example, [20,28]). This natural association of temperature and light-dependent phase resetting likely facilitates cooperative entrainment. As shown here, separate or combined treatments with daily light/dark cycles and temperature gradients produce similar circadian behavioral phases when the phases of ascending and descending temperature match those of light and dark, respectively. In this arrangement, as in natural conditions, the daily temperature minimum coincides with the time just before dawn, while the temperature maximum is associated with a time point in the second half of the day.

What might be the molecular mechanism underlying the circadian temperature synchronization described in this study? Analysis of temperature-entrained gene expression indicates that the phase relationship between behavioral circadian rhythms and molecular rhythms in the fly head is relatively stable and similar to that observed previously for light entrainment. Peak expression of CLK/CYC regulated transcripts (per, tim, vri) occurs shortly after peak temperature and PER and TIM protein expression show the expected ~6-h delay relative to their transcript levels [29]. These observations confirm and complement analyses from a previous study comparing the genome-wide transcript profiles in the adult head during and after treatment of wild-type and arrhythmic mutant flies with daily light/dark or square-wave temperature cycles [21]. In that study many circadian transcript profiles were identified that showed the same fixed relationship between light-entrained and temperature-entrained circadian phases, indicating that the same molecular clock mechanism is responsible for both light-entrained and temperature-entrained gene expression rhythms. Moreover, the expression levels of clock gene transcripts (per, tim, Clk, vri, Pdp1-ε, cry) and proteins (PER, TIM) were found to respond to environmental temperature changes in a clock-dependent manner [21,30]. The identification here of the peak temperature phase as the main determinant of the temperature-entrained behavioral phase suggests that associated molecular events are responsible for temperature entrainment. Perhaps the two most relevant of these are the peak in transcript levels for CLK/CYC-regulated transcripts, including per and tim, and the accelerated accumulation of PER and TIM proteins. Temperature-dependent modulation of circadian phase has been related to changes in per transcript levels as well as PER and TIM protein levels [16,21,24,26,30-34].

As mentioned before, per transcript levels are known to be regulated by temperature-dependent splicing of the terminal intron of per and this, in turn, affects PER protein levels as well as locomotor activity in the evening [16,24]. At lower constant temperatures splicing of this intron is enhanced, which leads to earlier accumulation of per mRNA and protein and advanced evening activity. This mechanism does, however, not appear to be required for synchronization of the clock to daily temperature cycles. Entrainment of circadian behavior to daily temperature cycles presented in constant darkness was largely unaffected in mutants that prevent splicing of the terminal per intron (Additional file 6). In addition, per splicing levels in wild-type flies did not show obvious daily changes in temperature cycles during constant light [26].

Complex formation of PER and TIM protein with CRYPTOCHROME (CRY) [34] and destabilization of TIM and PER protein [32] have been implicated in circadian phase resetting in response to heat shock treatment. However, these responses can be separated from entrainment to daily temperature cycles based on the requirement of the former for treatment with high temperatures (>34°C) and the involvement of CRY [32,34] which are both dispensable for synchronization to daily temperature cycles [19,21,25,26,33,34]. Moreover, heat shock treatment preferentially results in phase delays, whereas both phase advances and delays are readily observed in response to daily temperature cycles ([25,34]; data not shown).

Changes in TIM protein levels in a subset of clock neurons (type 2 Dorsal Neurons, DN2s, and Lateral Posterior Neurons, LPNs, and, to a lesser extent, type 1 and 3 Dorsal Neurons, DN1s and DN3s) were reported in association with a shifted alignment of simultaneously applied light/dark and temperature cycles [31]. It is not yet clear, however, whether these changes could be responsible for the advanced onset of evening activity recorded when the thermophase and cryophase of a square-wave temperature cycle were centered around the lights-on and lights-off transitions, respectively [31]. Observations resulting from the combination of a light/dark cycle with an anti-phase daily temperature gradient described here, for wild-type, but not arrhythmic mutant flies, indicate that the advance in evening activity onset is a clock-dependent response rather than a startle response following a sudden temperature decrease. Apart from a possible temperature-specific role for the LPNs, the set of clock neurons responsible for circadian temperature entrainment mostly coincides with the set known to control circadian light entrainment [25]. In this context, the subtle changes in TIM expression in the DN1s, which have been reported to modulate evening activity in the context of light treatment [35-37], may merit further examination.

Thus, although there is precedent for the temperature-dependent regulation of circadian phase via modulation of either per transcript levels or PER and TIM protein levels, neither of these mechanisms has been conclusively shown to account for circadian synchronization to environmental temperature cycles. Further molecular studies tracking the dynamics of circadian transcript and protein expression as well as post-translational modifications during temperature cycle-dependent phase advances and delays are expected to provide insight into this issue.

In contrast to Drosophila and other poikilothermic organisms, homeothermic mammals such as humans do not have a body temperature that directly tracks environmental temperature. Nevertheless, there is evidence supporting the involvement of daily temperature cycles in the maintenance of mammalian circadian rhythms because body temperature itself not only exhibits daily clock-controlled rhythms but can also act as a synchronizing cue [38,39]. Two pathways in particular have been proposed to be involved in mediating entrainment to mammalian body temperature cycles: (1) Heat Shock Factor 1 (HSF1)-dependent transcriptional regulation and (2) control of gene expression by cold-induced RNA binding proteins [39]. In this context, it is intriguing that rhythmic expression of HSF1-regulated heat shock proteins genes was not only observed in the mouse liver [40], but also in the heads of Drosophila exposed to a synchronizing daily temperature cycle of modest amplitude [21]. Additional research is needed to address the potentially conserved role of these genes in temperature entrainment.

Behavioral analysis

Individual flies were monitored in glass tubes on standard sugar agar media (5% sugar, 1% agar plus 0.07% Tegosept [Genesee Scientific, San Diego, CA] to inhibit fungal growth) and their locomotor activity was analyzed with the Drosophila Activity Monitoring System (DAMSystem; TriKinetics, Waltham, MA) housed in Percival I-36VL incubators, Percival Scientific, Perry, IA) equipped with fluorescent lights, humidity control, and Advanced Intellus Control systems to generate ramping temperature profiles. Unless otherwise specified, temperature entrainment and free running analyses were conducted in constant darkness at 70% relative humidity. Individual and median activity records, as well as periodic activity profiles, time series, and periodograms were generated using ClockLab Software (ActiMetrics, Wilmette, IL). Median activity records were created from the raw individual activity records on a point-by-point basis without prior normalization. Activity profiles representing the average of the median activity records (± S.E.M.) across the included intervals of 24 h or the intrinsic circadian period length τ were generated from flies stably synchronized to an environmental protocol or under free running conditions, respectively. Data from days spanning the transition from synchronizing to free running conditions or prior to stable synchronization were excluded from the activity profile analyses. Intrinsic circadian period length τ was determined by conducting a chi-square periodogram analysis of median activity data using 0:00 am on the first full day of free running conditions (as detected by the DAMSystem software) as starting point. The average activity profiles during synchronizing conditions were plotted relative to the following reference points at t = 0: DAMSystem time 0:00 am (Figure ​(Figure1,1, left-hand diagrams; Figure ​Figure2C,2C, left-hand and right-hand diagrams; Figure ​Figure3;3; top diagram in Additional file 1), 6:00 am (Figure ​(Figure5B5B and ​and5C;5C; Additional file 2), and 9:00 pm (Figure ​(Figure2C,2C, center diagrams). The average activity profiles during free running conditions were plotted relative to 0:00 am (DAMSystem time) on the first full day of free run with the following shifts in circadian phase angle at t = 0: unshifted (Figure ​(Figure1,1, right-hand diagrams; Figure ​Figure4B4B left-hand and right-hand diagrams; Figure ​Figure8D8D top diagrams; bottom diagram in Additional file 1), 90° advance (Figure ​(Figure5A;5A; Figure ​Figure6B6B and ​and6C;6C; Figure ​Figure8B8B top diagrams), 45° delay (Figure ​(Figure4B4B center diagrams), 90° delay (Figure ​(Figure8B8B bottom diagrams), and 180° change (Figure ​(Figure8D8D bottom diagrams). The phase of the peak and offset of activity during and following exposure to daily environmental cycles was determined from the activity profiles by interpolation as illustrated by Additional file 1. Because the activity profiles for free running data do not represent the time from the onset of free run during the day of transition and 0:00 am (DAMSystem time) on the first full day of free run, a correction had to be used to calculate free running phases relative to the previous environmental cycle. Specifically, if the flies were under free-running conditions for n hours on the day that the transition from entrainment to free run took place and they showed a subsequent free running period length τ, a phase correction of [n × (24 – τ)]/24 h of circadian time was applied. These corrections have been incorporated in Figures ​Figures4A,4A, ​,6A,6A, ​,8A8A and ​and8C8C as well as Additional files 3 and 7. Numbers of flies (n) and days (d) used to generate median activity records and activity profiles are indicated in Figure ​Figure1,1, and Additional files 3 and 7, or as follows.

Northern blot analysis

Total RNA was extracted from approximately 100 μl of adult heads per time point using guanidinium thiocyanate followed by centrifugation in Cesium chloride solution as previously described [41]. Northern analysis was performed according to published protocols [21]. Radioactive signals on the blots were visualized and quantitated with a Storm 840 Phosphorimager (GE healthcare, Piscataway, NJ) and the results plotted in Microsoft Excel (Microsoft Corporation, Redmond, WA).

Western blot analysis

Total protein was extracted from adult heads and subject to Western blotting analysis with anti-ACTIN (Sigma A 2066), Sigma-Aldrich (St Louis, MO) anti-PER, and anti-TIM antisera as previously described [21]. Specificity of the detected PER and TIM protein signals was verified by inclusion of an extract from y per01w; tim01 flies on the same blot (not shown).

Abbreviations

Authors' contributions

HW conceived of the study, designed the experiments, carried out behavioral studies and data analysis, and drafted the manuscript. JC contributed to behavioral and Northern analyses. TG conducted Western analyses. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1:

Figure S1. Determination of peak and offset of locomotor activity. In this example the 12 female Canton-S flies from Figure ​Figure11 are used as an example. First individual activity records were compared for uniformity to ensure that the generation of median data did not obscure the presence of strong divergent trends in subsets of individuals. Next, median locomotor activity was determined. The daily activity profile representing temperature entrainment was generated by averaging median daily activity for the experimental days following stable synchronization of behavior and then plotting mean ± S.E.M. activity relative to a 24-h period. A modified procedure was used to generate the daily activity profile representing free running conditions. In this case the presence of significant circadian rhythmicity during the free running days immediately following entrainment was tested by chi-square periodogram analysis (using the CLOCKLAB software package). If significant circadian periodicity (P < 0.01) was detected, median daily activity was averaged for the specified circadian period length and plotted as mean ± S.E.M. activity relative to the circadian period (τ). The phases of daily activity peak and offset were determined using interpolation. The phase interval during which the (mean + S.E.M.) activity value was equal or higher than highest mean activity value observed was used to define the peak (as the mid-point of this interval). The phase interval following the peak during which the half-maximal activity value (50% of the maximal (mean + S.E.M.) value) fell within the observed mean ± S.E.M. activity range was used to define the offset (as the mid-point of this interval). In case of bimodal profiles the evening activity peak was used for this analysis.

Figure S2. Daily temperature gradient protocols used to identify the phase determinants of circadian temperature entrainment. The graphs represent the experimental temperature protocols used in the analyses of Figures ​Figures22 and ​and4.4. These protocols are depicted during the last day of entrainment to one of eight daily temperature gradients, a day of transition, and the first day of free running conditions at constant temperature, all carried out in constant darkness. Note that there were eight different entrainment profiles (Z1-8; represented in Figure ​Figure2A2A and ​and2B)2B) and four different free running conditions at constant temperature (C1-4) that were used together in 12 different combinations (represented in Figure ​Figure4A).4A). Because two pairs of protocols (Z7T1C2 and Z7T2C2; Z7T3C4 and Z7T4C4) combined the same entrainment and free running conditions, but differed in the daily timing of the transition into free run the total number of different protocols was 14. Temperature ranges during entrainment: 14 to 22.5°C (Z1), 17 to 25°C (Z2), 18 to 25°C (Z3, Z4, Z5, Z6), 16 to 28°C (Z7), and 22 to 30°C (Z8). Maximal rate of change during entrainment: 1.4°C/h (Z1), 1.3°C/h (Z2), 1.75°C/h (Z3, Z6), 0.6°C/h (Z4), 0.8°C/h (Z5), and 1°C/h (Z7, Z8). Free running temperatures: 17°C (C1), 18°C (C2), 22°C (C3), 25°C (C4). For profiles Z3C2, Z6C4, and Z4C2, the conditions during the transition days were identical to those of free running conditions. Additional file 3 contains the incubator programs used to generate each protocol as well as behavioral data representing each protocol or combination of protocols.

Table TS1. This table contains the experimental data represented in Figures ​Figures2A2A and ​and2B2B and ​and4A4A (as indicated by color coding) as well as detailed information for each experimental condition regarding the temperature protocols, number and gender of flies, free running period length (tau), and estimated peak and offset phases during entrainment and free running conditions. The indicated corrected free running phases reflect adjustments for free running intervals occurring on the day of transition that were not accounted for in the activity profiles (see Methods).

Figure S3. Daily temperature gradients produce strong clock-independent behavioral responses even in the presence of light/dark cycles. The six panels with daily activity profiles in this figure represent the mean ± S.E.M. during 12-h light/12-h dark conditions at constant 25°C (gray shading) or 12-h light/12-h dark conditions in the presence of a daily 28°C to 16°C to 28°C temperature gradient (black shading) as determined from median activity records of y per0w, y w; tim01, and y w;; cyc01 flies. In the presence of a light/dark cycle at constant temperature per01 and tim01 flies showed startle responses to both light/dark transitions, while cyc01 flies exhibited a reduced startle response that was detected only at the lights-off transition. In addition, cyc01 males showed paradoxical masking (that is, increased activity during the dark) under these conditions. Consistent with their reduced light sensitivity and robust temperature-driven responses, cyc01 flies showed activity profiles that tracked daily temperature gradients even in the presence of light/dark cycles. The activity profiles of per01 and tim01 flies in the presence of the combined light and temperature protocol were similar, but contained a number of additional light-dependent features (cf. black traces with per01 and tim01 profiles in Figure ​Figure3):3): First, the per01 and tim01 activity profiles under these conditions showed a more gradual decrease in activity in the presence of the anti-phase light/dark cycle than in constant darkness, second, per01 and tim01 males showed an acute and temporary light-dependent down-regulation of activity after the lights-on transition (as indicated by '*'), and, third, the tim01 flies showed suppressed activity for 5 to 6 h following the lights-off transition (as indicated by '#').

Figure S4. Composite temperature profiles combining both daily temperature gradients and continuous increases or decreases. The left and right panels illustrate how the composite temperature profiles used for the experiments in Figure ​Figure77 resulted from combinations of daily temperature gradients with gradual 3°C temperature increases or decreases, respectively. The profiles in the first row represent combinations with constant temperature, whereas the profiles in the second and third rows involved daily temperature gradients with a 1.5°C or 4°C range, respectively.

Figure S5. The phase of temperature-entrained circadian behavior is modulated by the relative temperature of release into free run in the absence of thermosensitive splicing of the terminal intron of the per gene. The introduction of mutations that block temperature-dependent splicing of per did not prevent temperature-dependent modulation of the circadian phase of locomotor activity in male flies. Female circadian behavior appeared to be largely insensitive to the relative temperature during free running conditions, but this appeared to be due to the genotype used for transgenic rescue of the per01 mutation and unrelated to thermosensitive splicing of per. Temperature-dependent circadian locomotor behavior was determined for flies in which the per01 mutation was rescued by transgenes containing either wild-type genomic sequence (perG) or modified alleles that lack (perB') or constitutively maintain the terminal intron (perA), such that splicing at this intron was blocked. Following entrainment to a daily temperature gradient (ranging between 17°C and 25°C in constant dark (DD) conditions) flies were released under free running conditions at constant peak (25°C) or trough temperature (17°C) conditions. The diagrams represent median locomotor activity profiles relative to the intrinsic circadian period, tau. The dotted lines inside as well as the shading in the horizontal bars below the activity profiles represent the previous daily temperature gradient.

Table TS2. This table contains the experimental data represented in Figure ​Figure8A8A and ​and8C,8C, as well as detailed information for each experimental condition regarding the light and temperature protocols, number and gender of flies, free running period length (tau), and estimated peak and offset phases during entrainment and free running conditions. The indicated corrected free running phases reflect adjustments for free running intervals occurring on the day of transition that were not accounted for in the activity profiles (see Methods). Only the 'step up' and 'step down' protocols included an interval (12 h) of unaccounted free run on the transition day. No correction was required for the 'ramp' and 'zigzag' protocols.

Acknowledgements

We thank Sara Freeman, Claire Olsen, Rachel Siegmund, and Nella Solodukhina for technical support, Sara Freeman, Nick Hargus, Laura Straub, and Ashli Moore for contributions to behavioral analyses, Whitney Nelson for contributions to Western analysis, Michael Young, Jeff Hall, and Michael Rosbash, for sharing antibodies and fly stocks, and Jay Hirsh, Michael Menaker, Carla Green, and Ignacio Provencio for helpful discussions and advice. HW, JC, and TG were supported by grant R01 GM78339 from the National Institutes of Health to HW and a Distinguished Young Investigator award from the University of Virginia to HW.